The present invention relates to a lytic reagent composition for determination of nucleated blood cells in a blood sample. More specifically the lytic reagent composition enables differentiation of nucleated red blood cells from other cell types in a blood sample by a direct current impedance measurement. In addition, the lytic reagent composition can further be used for measuring total hemoglobin concentration of the blood sample.
Normal peripheral blood contains mature red blood cells which are free of nucleus. Nucleated red blood cells (NRBCs), or erythroblasts, are immature red blood cells. They normally occur in the bone marrow but not in peripheral blood. However, in certain diseases such as anemia and leukemia, NRBCs also occur in peripheral blood. Therefore, it is of clinical importance to measure NRBCs. Traditionally, differentiation and enumeration of NRBC are performed manually. The process involves the smearing of a blood sample on a microscope slide and staining the slide, followed by manual visual analysis of the individual slide. The NRBC concentration is reported as numbers of NRBC per 100 white blood cells. Usually, 200 white blood cells and the numbers of NRBC present in the same region on a blood smear are counted and the numbers are divided by 2 to express the NRBC concentration as the numbers of NRBC/100 WBC. This approach is extremely time-consuming as well as being subjective to the interpretation of the individual analyzing the slide.
In recent years, several fluorescence flow cytometry methods have been developed for differentiating NRBCs. These methods utilizes specific nuclear staining technique to distinguish NRBCs from other cell types because it is difficult to differentiate NRBCs based on their electronic or optical properties.
U.S. Pat. No. 5,298,426 (to Inami et al.) discloses a fluorescence method for differentiating NRBCs. The method utilizes a two-step staining using a first fluid which is an acidic hypotonic fluorescent dye solution, and a second fluid which changes the osmolality and pH of the first fluid. Inami et al. teaches that the first fluid contains an erythroblast-staining dye that diffuses into nucleated red blood cells to specifically stain their nuclei, and then separating a group of NRBCs from other cell groups on a two-dimensional plot whereby the results of NRBC differentiation are computed.
U.S. Pat. Nos. 5,516,695 and 5,648,225 (to Kim et al) disclose a multipurpose lysing reagent system and a method of use for subclassification of nucleated blood cells. The lysing reagent comprises a non-quaternary ammonium salt, an aliphatic aldehyde, a non-phosphate buffer which is inert to the aliphatic aldehyde, and a nuclear stain. The method comprises the steps of lysing a blood sample with the lysing reagent, incubating the sample mixture at an elevated temperature, and determining the nucleated blood cells including NRBCs with an automated electro-optical hematology instrumentation.
U.S. Pat. No. 5,559,037 (to Kim et al) discloses a method for flow cytometric analysis of NRBCs and leukocytes. The method comprises lysis of red blood cells and NRBC cytoplasm from a whole blood sample to expose the NRBC nuclei to a vital nuclear stain and minimizing the permeation of the vital nuclear stain into the leukocytes and analyzing the sample by measuring fluorescence and two angles of light scatter. Since leukocytes are also nucleated cells, staining of these cells needs to be prevented to avoid interference to the fluorescence measurement. The preservation of leukocyte membrane and minimizing the permeation of the nuclear stain into the leukocytes are achieved by concurrently fixing the leukocytes with an aliphatic aldehyde during lysis of red blood cells. The aldehyde fixatives are known as hazardous chemicals. In addition, the method requires heating of the reagent to 42xc2x0 C. in order to obtain the NRBC and leukocyte differentiations.
EP 1 004 880 A2 discloses reagents and a method for discrimination and counting of nucleated red blood cells. The reagents include a hemolytic agent for dissolving red blood cells, and conditioning white blood cells and NRBCs in a sample to be suitable for staining; and at least one fluorescent dye selected to stain white blood cells and NRBCs differentially. The method includes the steps of lysing red blood cells, staining white blood cells and NRBCs, assaying the sample by measuring at least one scattered light parameter, and at least one fluorescence parameter.
U.S. Pat. No. 5,874,310 (to Li et al) discloses reagents and a method for differentiation of nucleated red blood cells. The method includes exposing a blood sample to a lysing reagent system to lyse mature red blood cells and analyzing the sample in a flow cell by two low angle light scatter measurements to differentiate NRBCs from other cell types. The method further includes a concurrent differentiation of white blood cells using electronic and optical analysis, wherein the electronic analysis is a DC impedance measurement.
U.S. Pat. No. 5,917,584 (to Li et al) discloses a method for differentiation of nucleated red blood cells. The method includes lysing mature red blood cells in a blood sample; analyzing the sample in a flow cell by two angles of light scatter measurement to differentiate NRBCs from other cell types, wherein the second light scatter signal is a medium angle or a right-angle light scatter signal.
The above described methods enable differentiation and enumeration of NRBCs and leukocytes by fluorescence flow cytometry and light scatter measurements. However, fluorescence and light scatter measurements are complex and expensive detection methods.
Many current non-fluorescence automated hematology analyzers, such as Abbott Cell-Dyn(copyright) 3500, COULTER(copyright) Gen*S(trademark), Bayer Advia*120(copyright), and Sysmex(trademark) NE-9000 are only able to provide a NRBC flagging for the possible presence of NRBCs in an analyzed blood sample when the instruments sense an increased amount of signals near red blood cell debris area of an obtained cell distribution histogram. However, such methods are prone to generate false positive flaggings because many other blood abnormalities can cause increased signals at the same area, such as platelet clumps and sickle cells, as well as red cell debris from insufficiently lysed blood samples. In these methods NRBCs are not distinctly identified. Instead, only a common NRBC sample distribution pattern in a histogram or a dotplot is recognized by the instrument which can be confused with a similar pattern generated by above-mentioned other causes. For the flagged samples, including false positive flags, re-examination of the sample with manual method is required in clinical laboratories.
Furthermore, a well known problem with NRBC containing samples is erroneous white blood cell count (WBC) reported by hematology analyzers on these samples. Since the nuclear volumes of NRBCs are close to those of white blood cells, the NRBCs are commonly counted as white blood cells on hematology analyzers which measure the sizes of blood cells, resulting an elevation of the WBC. Therefore, correction of NRBC contribution to the WBC reported from hematology analyzer is required for samples containing NRBC. Current practice in clinical laboratory is to subtract the numbers of NRBC obtained by manual count from the WBC reported by the hematology analyzers. This is time consuming and error prone.
In a different aspect, various lytic reagent compositions for analysis of white blood cells are known in the art. U.S. Pat. No. 5,618,733 (to Tsuji et al) discloses a reagent for analyzing leucocytes, which comprises an ionic surfactant; at least one organic compound having a hydrophobic group and an acidic group which has a negative charge in an aqueous solution for preserving leukocyte morphology by combining with a cationic component in leukocytes; a nonionic surfactant; and a buffer.
U.S. Pat. No. 4,528,274 (to Carter et al) discloses a lytic reagent for determination of at least two leukocyte populations in blood. The lytic reagent comprises an aqueous solution of at least two quaternary ammonium salts, and one non-cationic surfactant additive which includes nonionic polyoxyethylated alkylphenol. The prior art teaches that the quaternary ammonium salts and the additive are in sufficient amounts for positioning white blood cell populations relative to one another within the measurement time of the blood analyzer.
These lytic reagents enable differentiation of white blood cell subpopulations by preserving the morphology of white blood cells, or by controlling the size of the subpopulations. However, these reagents do not differentiate nucleated red blood cells from other cell types.
Furthermore, measurement of hemoglobin (Hgb) concentration of blood samples is an integral part of blood analysis, which is important for disease diagnosis and for monitoring responses to medical treatment. It is desirable to be able to accomplish multiple diagnostic analyses such as enumerating nucleated blood cells and measuring hemoglobin concentration of a blood sample using the same reagent and concurrent detections.
Among the many well known methods for hemoglobin determination, the cyanmethemoglobin method has been recommended as a standard by the International Committee for Standardization in Hematology. However, the presence of cyanide in the reagent waste has caused enormous environmental concern. In last ten years, a tremendous effort has been given to develop automated hemoglobin analysis methods without utilizing cyanide.
U.S. Pat. No. 5,242,832 (to Sakata) discloses a cyanide-free lysing reagent for counting white blood cells and measuring the hemoglobin concentration in a blood sample. The lysing reagent comprises at least one first surfactant which is a quaternary ammonium salt, at least one second surfactant which includes cationic and amphoteric surfactants, and at least one hemoglobin stabilizer selected from the group including Tiron, 8-hydroxyquinoline, bipyridine, 1-10-phenanthroline, phenolic compounds, bisphenol, pyrazole and derivatives, second phenyl 5-pyrazolone and derivatives, phenyl 3-pyrazolone, and imidazole and its derivatives.
PCT/US95/02897 (to Kim) discloses a cyanide-free method and reagent for determining hemoglobin in a whole blood sample. The reagent comprises a ligand selected from the group consisting of imidazole and derivatives, N-hydroxyacetamide, H-hydroxylamine, pyridine, oxazole, thiazole, pyrazole, pyrimidine, purine, quinoline and isoquinoline, and a surfactant with a strong erythrolytic capability selected from the group consisting of lauryl dimethylamine oxide and octylphenoxy polyethoxyethanol. The analysis method is fast, less than 10 seconds. However, the reagent only performs under an extreme alkaline condition, pH from 11 to 14. In addition, no capability of counting leukocytes, nor differentiating nucleated red blood cells from other cell types is taught by Kim.
U.S. Pat. No. 5,763,280 (to Li et al) discloses cyanide-free reagents for measuring hemoglobin in a blood sample, counting leukocytes, and differentiating leukocyte subpopulations. The reagent comprises a hemolytic surfactant selected from the group consisting of quaternary ammonium salts, pyridinium salts, organic phosphate esters, and organic sulfonates to lyse erythrocytes and release hemoglobin, and an organic ligand selected from the group consisting of triazole and its derivatives, tetrazole and its derivatives, alkali metal salts of oxonic acid, melamine, aniline-2-sulfonic acid, quinaldic acid, 2-amino-1,3,4-thiadiazole, triazine and its derivatives, urazole, DL-pipecolinic acid, isonicotinamide, anthranilonitrile, 6-aza-2-thiothymine, adenine, 3-(2-thienyl)acrylic acid, benzoic acid and alkali metal and ammonium salts of benzoic acid, and pyrazine and its derivatives to form a stable chromogen with hemoglobin.
U.S. Pat. No. 5,882,934 (to Li et al) also discloses cyanide-free reagents for measuring hemoglobin in a blood sample, counting leukocytes, and differentiating leukocyte subpopulations. The reagents further comprise salts, in addition to hemolytic surfactants and organic ligands, for adjusting conductivity of the reagents for impedance measurement. However, none of the above described cyanide-free reagent and hemoglobin measurement methods enables differentiation of nucleated red blood cells from other cell types, and enumeration of nucleated red blood cells in a blood sample.
Based on foregoing, there exist a need for a reagent that enables simple and less costly analysis method for differentiating and enumerating nucleated red blood cells. Furthermore, it is desirable to have a multifunctional reagent enabling enumeration of nucleated blood cells, differentiation of nucleated red blood cells from other cell types, and measurement of hemoglobin concentration in the absence of cyanide.
The present invention relates to a lytic reagent composition for lysing red blood cells, and measuring nucleated blood cells in a blood sample. The lytic reagent composition comprises at least one quaternary ammonium surfactant, an ethoxlyated phenol, and an ethoxylated alcohol, and having a pH ranging from about 2 to about 11. When mixed with a blood sample prediluted by a diluent, the lytic reagent composition lyses red blood cells and enables a differentiation of nucleated red blood cells from other cell types by DC impedance measurement.
The lytic reagent composition can further comprise an organic ligand in a sufficient amount to form a chromogen with hemoglobin for determining total hemoglobin concentration of a blood sample by measuring spectrophotometric absorbance at predetermined wavelengths.
The present invention further relates to a lytic reagent system for measuring nucleated blood cells in a blood sample, which comprises a lytic reagent composition and a diluent. The lytic reagent composition comprises at least one quaternary ammonium surfactant, an ethoxlyated phenol, and an ethoxylated alcohol, and having a pH ranging from about 2 to about 11. The diluent is a neutral aqueous solution comprising a salt or salts to adjust conductivity of the diluent sufficient for an impedance measurement, and an antimicrobial agent. The lytic reagent system can further comprise an organic ligand either in the lytic reagent composition or in the diluent for measuring hemoglobin concentration of a blood sample.
In a further embodiment, the present invention also relates to a lytic reagent composition comprising at least one quaternary ammonium surfactant, an ethoxlyated phenol, an ethoxylated alcohol, and a salt or salts to adjust the conductivity of the lytic reagent composition sufficient for impedance measurement. The lytic reagent composition can further comprise an organic ligand for hemoglobin measurement. This lytic reagent composition can be used for a combined diluting and lysing, and preparing a blood sample for measuring nucleated blood cells in one step.